In high risk infants, wheezing-associated illness with rhinovirus (RV) is the most significant risk factor for asthma development, more so than allergen sensitization or infection with respiratory syncytial virus (RSV). Thus, RV infection in early life in combination with other factors such as genetic background, allergen exposure and microbiome, may modulate the immune response, increasing the likelihood of asthma development. To test this, we developed a neonatal mouse model of RV1B infection. In contrast to mature animals, 6 day-old mice infected with RV1B develop sustained airways hyperresponsiveness, mucous metaplasia and IL-13 production. This asthma-like phenotype is dependent on a population of IL-13-producing type 2 innate lymphoid cells (ILC2s). In this project, we will test the general hypothesis that, in susceptible individuals, early-life RV infection contributes to childhood asthma development via the expansion of IL-13-producing ILC2s. To test this general hypothesis, three specific aims are proposed: Specific Aim 1. Determine the roles of TSLP and IL-33 in RV-induced mucous metaplasia and airways hyperresponsiveness in neonatal BALB/c mice. We hypothesize that, in 6 day-old immature mice: i) RV infection increases epithelial cell production of TSLP and IL-33; ii) TSLP and IL-33 are required for maximal mucous metaplasia and airways hyperresponsiveness; and iii) RV-induced TSLP production is permitted by a deficient IFN-? response. Specific Aim 2. Determine the contribution of ILC2s to RV-induced airway responses. We hypothesize that: i) in immature mice, RV-induced TSLP, IL-25 and IL-33 regulate expansion and IL-13 production by ILC2s; (ii) ILC2s are required and sufficient for maximal RV-induced type 2 cytokine expression, mucous met- aplasia and airways hyperresponsiveness; and 3) IFN-? attenuates ILC2 expansion and IL-13 production. Specific Aim 3. Determine the effects of neonatal RV infection on responses to subsequent heterologous re-infection. We hypothesize that: i) RV infection of immature mice regulates the immune response to future viral infections, leading to type 2 rather than type 1 responses; ii) synergisti type 2 responses are driven by ILC2s. For Aims 1-3, to determine whether ILC2s constitute a common cellular response to early-life respiratory viral infection, we will compare RV1B, RV2 and RSV-A infections in 6 day-old immature mice and 8 month-old mature mice. Also, to support Aims 1 and 2, we will analyze TSLP, IL-25 and IL-33 levels and ILC2s in nasal and tracheal lavage samples taken from infants hospitalized with acute respiratory viral infections. Finally, to begin to understand why only some infants exposed to early-life viral infection may develop asthma, we will perform proof-of-concept experiments examining mucous metaplasia, airways hyperresponsiveness and ILC2s in RV-infected C57BL/6 mice and germ-free mice.